Parmodulins Inhibit Thrombus Formation Without Inducing Endothelial Injury Caused by Vorapaxar

Protease-activated receptor-1 (PAR1) couples the coagulation cascade to platelet activation during myocardial infarction and to endothelial inflammation during sepsis. This receptor demonstrates marked signaling bias. Its activation by thrombin stimulates prothrombotic and proinflammatory signaling, whereas its activation by activated protein C (APC) stimulates cytoprotective and antiinflammatory signaling. A challenge in developing PAR1-targeted therapies is to inhibit detrimental signaling while sparing beneficial pathways. We now characterize a novel class of structurally unrelated small-molecule PAR1 antagonists, termed parmodulins, and compare the activity of these compounds to previously characterized compounds that act at the PAR1 ligand–binding site. We find that parmodulins target the cytoplasmic face of PAR1 without modifying the ligand-binding site, blocking signaling through Gαq but not Gα13 in vitro and thrombus formation in vivo. In endothelium, parmodulins inhibit prothrombotic and proinflammatory signaling without blocking APC-mediated pathways or inducing endothelial injury. In contrast, orthosteric PAR1 antagonists such as vorapaxar inhibit all signaling downstream of PAR1. Furthermore, exposure of endothelial cells to nanomolar concentrations of vorapaxar induces endothelial cell barrier dysfunction and apoptosis. These studies demonstrate how functionally selective antagonism can be achieved by targeting the cytoplasmic face of a G-protein–coupled receptor to selectively block pathologic signaling while preserving cytoprotective pathways

A New Pathway for Protein Haptenation by beta-Lactams

"This is the peer reviewed version of the following article: Pérez-Ruíz, Raúl, Emilio Lence, Inmaculada Andreu, Daniel Limones-Herrero, Concepción González-Bello, Miguel A. Miranda, and M. Consuelo Jiménez. 2017. A New Pathway for Protein Haptenation by β-Lactams. Chemistry - A European Journal 23 (56). Wiley: 13986 94. doi:10.1002/chem.201702643, which has been published in final form at https://doi.org/10.1002/chem.201702643. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving."[EN] The covalent binding of beta-lactams to proteins upon photochemical activation has been demonstrated by using an integrated approach that combines photochemical, proteomic and computational studies, selecting human serum albumin (HSA) as a target protein and ezetimibe (1) as a probe. The results have revealed a novel protein haptenation pathway for this family of drugs that is an alternative to the known nucleophilic ring opening of beta-lactams by the free amino group of lysine residues. Thus, photochemical ring splitting of the beta-lactam ring, following a formal retro-Staudinger reaction, gives a highly reactive ketene intermediate that is trapped by the neighbouring lysine residues, leading to an amide adduct. For the investigated 1/HSA system, covalent modification of residues Lys414 and Lys525, which are located in sub-domains IIIA and IIIB, respectively, occurs. The observed photobinding may constitute the key step in the sequence of events leading to photoallergy. Docking and molecular dynamics simulation studies provide an insight into the molecular basis of the selectivity of 1 for these HSA sub-domains and the covalent modification mechanism. Computational studies also reveal positive cooperative binding of sub-domain IIIB that explains the experimentally observed modification of Lys414, which is located in a barely accessible pocket (sub-domain IIIA).Financial support from Ministerio de Economia, Industria y Competitividad (CTQ2013-47872-C2-1-P, CTQ2016-78875-P, SAF2013-42899-R, SAF2016-75638-R), Instituto de Salud Carlos III (RD12/0013/0009 and RD16/0006/0030), Generalitat Valenciana (PROMETEOII/2013/005), Xunta de Galicia (Centro singular de investigacion de Galicia accreditation 2016-2019, ED431G/09) and European Union (European Regional Development Fund -ERDF) is gratefully acknowledged. E.L. thanks the Xunta de Galicia for a postdoctoral fellowship. We are grateful to the Centro de Supercomputacion de Galicia (CESGA) for use of the Finis Terrae II supercomputer. The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to ProteoRed PRB2-ISCIII and is supported by grant PT13/0001, of the PE I+D+i 2013-2016, funded by ISCIII and FEDER.Pérez-Ruiz, R.; Lence, E.; Andreu Ros, MI.; Limones Herrero, D.; González-Bello, C.; Miranda Alonso, MÁ.; Jiménez Molero, MC. (2017). A New Pathway for Protein Haptenation by beta-Lactams. Chemistry - A European Journal. 23(56):13986-13994. https://doi.org/10.1002/chem.201702643S13986139942356Van Boeckel, T. P., Gandra, S., Ashok, A., Caudron, Q., Grenfell, B. T., Levin, S. A., & Laxminarayan, R. (2014). Global antibiotic consumption 2000 to 2010: an analysis of national pharmaceutical sales data. The Lancet Infectious Diseases, 14(8), 742-750. doi:10.1016/s1473-3099(14)70780-7Elander, R. P. (2003). Industrial production of β-lactam antibiotics. 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Efficacy in asthma of once-daily treatment with fluticasone furoate: a randomized, placebo-controlled trial

<p>Abstract</p> <p>Background</p> <p>Fluticasone furoate (FF) is a novel long-acting inhaled corticosteroid (ICS). This double-blind, placebo-controlled randomized study evaluated the efficacy and safety of FF 200 mcg or 400 mcg once daily, either in the morning or in the evening, and FF 200 mcg twice daily (morning and evening), for 8 weeks in patients with persistent asthma.</p> <p>Methods</p> <p>Asthma patients maintained on ICS for ≥ 3 months with baseline morning forced expiratory volume in one second (FEV₁) 50-80% of predicted normal value and FEV₁reversibility of ≥ 12% and ≥ 200 ml were eligible. The primary endpoint was mean change from baseline FEV₁at week 8 in pre-dose (morning or evening [depending on regimen], pre-rescue bronchodilator) FEV₁.</p> <p>Results</p> <p>A total of 545 patients received one of five FF treatment groups and 101 patients received placebo (intent-to-treat population). Each of the five FF treatment groups produced a statistically significant improvement in pre-dose FEV₁compared with placebo (p < 0.05). FF 400 mcg once daily in the evening and FF 200 mcg twice daily produced similar placebo-adjusted improvements in evening pre-dose FEV₁at week 8 (240 ml vs. 235 ml). FF 400 mcg once daily in the morning, although effective, resulted in a smaller improvement in morning pre-dose FEV₁than FF 200 mcg twice daily at week 8 (315 ml vs. 202 ml). The incidence of oral candidiasis was low (0-4%) and UC excretion was comparable with placebo for all FF groups.</p> <p>Conclusions</p> <p>FF at total daily doses of 200 mcg or 400 mcg was significantly more effective than placebo. FF 400 mcg once daily in the evening had similar efficacy to FF 200 mcg twice daily and all FF regimens had a safety tolerability profile generally similar to placebo. This indicates that inhaled FF is an effective and well tolerated once-daily treatment for mild-to-moderate asthma.</p> <p>Trial registration</p> <p><a href="http://www.clinicaltrials.gov/ct2/show/NCT00398645">NCT00398645</a></p

Design, analysis, and presentation of crossover trials

OBJECTIVE: Although crossover trials enjoy wide use, standards for analysis and reporting have not been established. We reviewed methodological aspects and quality of reporting in a representative sample of published crossover trials. METHODS: We searched MEDLINE for December 2000 and identified all randomized crossover trials. We abstracted data independently, in duplicate, on 14 design criteria, 13 analysis criteria, and 14 criteria assessing the data presentation. RESULTS: We identified 526 randomized controlled trials, of which 116 were crossover trials. Trials were drug efficacy (48%), pharmacokinetic (28%), and nonpharmacologic (30%). The median sample size was 15 (interquartile range 8-38). Most (72%) trials used 2 treatments and had 2 periods (64%). Few trials reported allocation concealment (17%) or sequence generation (7%). Only 20% of trials reported a sample size calculation and only 31% of these considered pairing of data in the calculation. Carry-over issues were addressed in 29% of trial's methods. Most trials reported and defended a washout period (70%). Almost all trials (93%) tested for treatment effects using paired data and also presented details on by-group results (95%). Only 29% presented CIs or SE so that data could be entered into a meta-analysis. CONCLUSION: Reports of crossover trials frequently omit important methodological issues in design, analysis, and presentation. Guidelines for the conduct and reporting of crossover trials might improve the conduct and reporting of studies using this important trial design

A Review of the Rationale for Additional Therapeutic Interventions to Attain Lower LDL-C When Statin Therapy Is Not Enough

Statins alone are not always adequate therapy to achieve low-density lipoprotein (LDL) goals in many patients. Many options are available either alone or in combination with statins that makes it possible to reach recommended goals in a safe and tolerable fashion for most patients. Ezetimibe and bile acid sequestrants reduce cholesterol transport to the liver and can be used in combination. Niacin is very effective at lowering LDL, beyond its ability to raise high-density lipoprotein and shift LDL particle size to a less atherogenic type. When statins cannot be tolerated at all, red yeast rice can be used if proper formulations of the product are obtained. Nutrients can also be added to the diet, including plant stanols and sterols, soy protein, almonds, and fiber, either individually or all together as a portfolio diet. A clear understanding of how each of these strategies works is essential for effective results

New Options in the Treatment of Lipid Disorders in HIV-Infected Patients

Since the introduction of HAART, there was a remarkably change in the natural history of HIV disease, leading to a notable extension of life expectancy, although prolonged metabolic imbalances could significantly act on the longterm prognosis and outcome of HIV-infected persons, and there is an increasing concern about the cardiovascular risk in this population. Current recommendations suggest that HIV-infected perons undergo evaluation and treatment on the basis of the Third National Cholesterol Education Program Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (NCEP ATP III) guidelines for dyslipidemia, with particular attention to potential drug interactions with antiretroviral agents and maintenance of virologic control of HIV infection. While a hypolipidemic diet and physical activity may certainly improve dyslipidemia, pharmacological treatment becomes indispensable when serum lipid are excessively high for a long time or the patient has a high cardiovascular risk, since the suspension or change of an effective antiretroviral therapy is not recommended. Moreover, the choice of a hypolipidemic drug is often a reason of concern, since expected drug-drug interactions (especially with antiretroviral agents), toxicity, intolerance, effects on concurrent HIV-related disease and decrease patient adherence to multiple pharmacological regimens must be carefully evaluated. Often the lipid goals of patients in this group are not achieved by the therapy recommended in the current lipid guidelines and in this article we describe other possibilities to treat lipid disorders in HIV-infected persons, like rosuvastatin, ezetimibe and fish oil